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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Two new phthalides with BuChE inhibitory activity from Ligusticum chuanxiong a
a
a
b
b
Jian Huang , Xiao-Qing Lu , Jin Lu , Guo-Yu Li , Hang-Yu Wang , a
c
ab
Li-Hong Li , Rui-Chao Lin & Jin-Hui Wang a
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, 110016, China b
School of Pharmacy, Shihezi University, Shihezi, 832002, China
c
National Institute for the Control of Pharmaceutical and Biological Products, Beijing, 100050, China Published online: 27 Nov 2013.
To cite this article: Jian Huang, Xiao-Qing Lu, Jin Lu, Guo-Yu Li, Hang-Yu Wang, Li-Hong Li, Rui-Chao Lin & Jin-Hui Wang (2013) Two new phthalides with BuChE inhibitory activity from Ligusticum chuanxiong, Journal of Asian Natural Products Research, 15:12, 1237-1242, DOI: 10.1080/10286020.2013.825610 To link to this article: http://dx.doi.org/10.1080/10286020.2013.825610
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Journal of Asian Natural Products Research, 2013 Vol. 15, No. 12, 1237–1242, http://dx.doi.org/10.1080/10286020.2013.825610
Two new phthalides with BuChE inhibitory activity from Ligusticum chuanxiong Jian Huanga, Xiao-Qing Lua, Jin Lua, Guo-Yu Lib, Hang-Yu Wangb*, Li-Hong Lia, Rui-Chao Linc and Jin-Hui Wangab* a
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School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China; bSchool of Pharmacy, Shihezi University, Shihezi 832002, China; c National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China (Received 6 May 2013; final version received 11 July 2013) Two new phthalides, chuanxiongdiolides A (1) and B (2), were isolated from the roots of Ligusticum chuanxiong Hort. Their structures were established by UV, IR, 1D (1H, 13C) and 2D (HSQC, 1H – 1H COSY, HMBC, NOESY) NMR, and HR-ESI-MS methods, and their absolute configurations were assigned via circular dichroism exciton chirality. The two compounds showed different degrees of inhibitory effects against butyrylcholine esterase. Keywords: Ligusticum chuanxiong; chuanxiongdiolide A; chuanxiongdiolide B; NMR; CD; anti-BuChE activity
1.
Introduction
The root of Ligusticum chuanxiong, a popular traditional Chinese medicine, has been widely used in the treatment of headache, rheumatic arthralgia, menstrual disorders [1], and swelling as well as pain due to traumatic injuries and liver diseases [2]. Previous reports on the biological components revealed the presence of dimeric phthalides [3]. Being interested in finding more biologically active substances from this folk medicine, we further undertook the investigation to explore their phytochemical composition. As a result, two new phthalides, chuanxiongdiolide A (1) and chuanxiongdiolide B (2), were isolated from the roots of L. chuanxiong. In this paper, we describe the structural elucidation of the two new compounds and their anti-butyrylcholine esterase (BuChE) activity (Figure 1).
2.
Result and discussion
Compound 1 was obtained as faint yellow oil (MeOH) with the molecular formula C24H32O4 as determined by HR-ESI-MS at m/z 385.2375 [M þ H]þ, indicating nine degrees at unsaturation. The IR spectrum exhibited the maximum absorptions at 1749 cm21 (carbonyl) and 1661 cm21 (olefin moieties). The 1H NMR spectrum of 1 showed one olefinic proton at dH 7.29 (1H, d, J ¼ 6.6 Hz, H-70 ) and two oxygensubstituted methine signals at dH 4.88 (1H, dd, J ¼ 7.4, 3.0 Hz, H-3) and 5.13 (1H, dd, J ¼ 10.3, 2.7 Hz, H-3 0 ). Twenty-four C-atoms signals including 2 CH3, 10 CH2, 6 CH, and 6 quaternary C-atoms were evident from the analysis of the 1 H, 13C NMR, and HSQC spectra. The 13 C NMR spectrum showed the resonances of one 5-membered unsaturated lactone ring at dC 82.0, 126.5, 169.2, and 173.8,
*Corresponding authors. Email: [email protected]; [email protected] q 2013 Taylor & Francis
1238
J. Huang et al. 10
1
8 4
1
7 O
O
6'
3' 7a' O
10 9
9'
10'
O
7' O 3'
3a' 4' 8'
1'
11
3a
H 7a'
5' 8'
O
8
3
5 6
HO
3a'
7'
7a
O
7a 5' 4'
O
4 3
7
6'
Downloaded by [Simon Fraser University] at 05:04 17 November 2014
9
3a
5 6
11
9'
11' 1
2
10'
11'
Figure 1. Chemical structures of 1 and 2 isolated from L. chuanxiong.
and the resonances of one a,b-unsaturated ester at dC 135.6, 141.8, and 167.2. Comparison of the 1H and 13C NMR spectra of 1 (Table 1) with those of the known compound (3Z0 )-(3a0 R,60 R,3R,6R,7R)-3,8dihydro-6.60 ,7.3a0 -diligustilide [3] revealed that the resonances of C-30 at dC 150.4 and C-80 at dC 108.8 disappeared, and the replacements of an oxygen-substituted methine at dC 84.1 and a methylene at dC 31.8 in 1 were observed, indicating that the double bond between C-30 and C-80 was saturated. Further analysis of the correlations in the 2D NMR spectra including 1 H – 1H COSY, HMQC, and HMBC (Figure 2) led to the planar structure of 1 as shown in Figure 1. The relative configuration of 1 was elucidated on the basis of the analysis of the NOESY correlations and molecular dynamics calibration with minimum energy by using Chem3D (Figure 2). In the NOESY spectrum, the cross-peaks between H-70 and H-4, H-70 and H-5, H-7 and H-40 , H-3 and H-30 , and H-6 and H-7 were observed. It was shown that only the configuration shown in Figure 2 could give such stereo structure correlations. The absolute configuration of 1 was determined by the circular dichroism (CD) exciton chirality method [4]. In the CD spectrum, it showed a clearly positive
cotton effect at 230 nm and a negative cotton effect at 215 nm due to the transition interaction between two a,bunsaturated lactones, indicating a positive chirality for 1 (Figure 4). So, the absolute configuration of 1 was determined as shown, which is named as chuanxiongdiolide A. Compound 2 was also a faint yellow oil. The molecular formula C24H32O5 was determined by the HR-ESI-MS at m/z 401.2331 [M þ H] þ , indicating nine degrees of unsaturation. The IR spectrum showed absorptions at 3429 cm 21 (hydroxyl), 1784, 1733 cm21 (carbonyl), and 1668 cm21 (olefin moieties). The 13C NMR spectrum also exhibited 24 C-atom signals which consisted of 2 CH3, 10 CH2, 5 CH, and 7 quaternary C-atoms in combination with careful analysis of the data of 1H and HSQC spectra (Table 1). Moreover, it showed the resonances of one 5-membered unsaturated lactone ring at dC 82.5, 123.3, 166.5, and 172.8; one ester group at dC 178.7; and two pairs of olefinic carbons at dC 116.4, 125.7, 131.0, and 146.0. The above data were found to display the main characteristics of dimeric phthalides. Besides, the 13C NMR spectrum showed two oxygen-substituted methines at dC 65.7 and 82.5. Furthermore, analysis of the unsaturation degrees
Journal of Asian Natural Products Research Table 1.
1
H (600 MHz) and 13C (150 MHz) NMR spectral data for compounds 1 and 2 in CDCl3. 1
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Position
1239
dC
1 3 3a 4
173.8 82.0 169.2 21.4
5
28.3
6 7 7a 8
38.7 38.9 126.5 30.9
9
26.1
10 11 10 30 30 a 40
22.3 13.6 167.2 84.1 47.8 26.0
50
25.2
60 70 70 a 80
40.2 141.8 135.6 31.8
90
28.4
100 110
22.1 13.7
2
dH (J in Hz)
dC
4.88 (1H, dd, J ¼ 7.4, 3.0) 2.07 (2H, m) 1.54 (1H, m) 1.85 (1H, m) 2.43 (1H, m) 2.94 (1H, br. d, J ¼ 8.8) 1.40 (1H, m) 1.86 (1H, m) 1.22(1H, m) 1.31(1H, m) 1.37(2H, m) 0.88 (3H, t, J ¼ 7.2)
1.39 (1H, m) 1.79 (1H, m) 1.29 (1H, m) 1.87 (1H, m) 2.97 (1H, dd, J ¼ 6.6, 2.4) 7.29 (1H, d, J ¼ 6.6) 1.57 (1H, m) 1.78 (1H, m) 1.48 (1H, m) 1.60 (1H, m) 1.31 (2H, m) 0.92 (3H, t, J ¼ 7.2)
O O HMBC
131.0 125.7 53.9 24.9
22.2 13.7 172.8 82.5 166.5 19.3 25.5 65.7 45.8 123.3 32.4
2.25 (1H, m) 2.47 (1H, m) 2.11 (1H, m) 2.27 (1H, m) 5.83 (1H, d, 9.7) 5.94 (1H, d, 9.7) 2.25 (1H, m) 2.31 (1H, m) 1.50 (2H, m) 1.33 (2H, m) 0.91 (3H, t, 7.2) 4.76 (1H, br.d, 6.2) 2.32 (1H, m) 2.38 (1H, m) 1.92 (1H, m) 2.22 (1H, m) 4.26 (1H, br. s) 2.98 (1H, br. s)
26.6
1.47 (1H, m) 1.88 (1H, m) 1.39 (2H, m)
22.5 13.9
1.35 (2H, m) 0.91 (3H, t, 7.2)
NMR spectra including 1H – 1H COSY, HMQC, and HMBC (Figure 3) deduced the gross structure of 2 as shown in
O
O
27.8
28.5
5.13 (1H, dd, J ¼ 10.3, 2.7)
inferred that the dimer was formed through one single bond of C-atoms. Careful analysis of the correlations in the 2D
178.7 146.0 116.4 18.8
dH (J in Hz)
NOESY
Figure 2. Key HMBC and NOESY correlations of 1.
1240
J. Huang et al. O O
HO
H
O O
HMBC
NOESY
Figure 1. Especially in the HMBC spectrum, the correlations from H-70 to C-3a and C-1, H-7 to C-70 , and H-60 to C7a verified that the two monophthalides were connected at C-7a and C-70 . The relative configuration of 2 was also determined via a NOESY experiment and molecular dynamics calibration with minimum energy by using the Chem3D (Figure 3), in which the correlations between H-30 and H-40 (dH 2.32), H-40 (dH 2.32) and H-50 (dH 1.92), H-50 (dH 1.92) and H-60 , and H-60 and H-70 were observed. Only the configuration shown in Figure 4 could give such stereo structure corre-
lations, indicating that the hydrogen bond between 60 -OH and 1ZCvO rendered the molecule in such stable configuration. In addition, the absolute configuration of 2 was determined by the CD exciton chirality method [4]. It showed a clearly positive cotton effect at 230 nm and a negative cotton effect at 215 nm due to the transition interaction between two excitons (Figure 4); so, the absolute configuration of 2 was determined as shown in Figure 1, which is named as chuanxiongdiolide B. BuChE was closely related with neurodegenerative diseases [5]. Therefore,
(+) (+)
∆ (M–1 cm–1)
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Figure 3. Key HMBC and NOESY correlations of 2.
3 2.5 2 1.5 1 0.5 0 –0.5 –1 –1.5 –2 190
2
1
Compound 1 Compound 2 240
290 Wavelength(nm)
340
Figure 4. CD spectra and exciton chirality of compounds 1 and 2.
390
Journal of Asian Natural Products Research anti-BuChE activity was evaluated for 1 and 2 in the concentration of 50 mM. The results showed that inhibitory rates of 1 and 2 were 63.0% and 21.7%, respectively, with the tetraisopropyl pyrophosphoramide (37.7%) as the positive control.
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3.
Experimental
3.1 General experimental procedures UV spectra were measured using the Shimadzu UV-2401 spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra were run on IRAffinity-1 FTIR spectrometer (Shimadzu). NMR spectra were obtained on Bruker AV-600 spectrometer (600 MHz for 1H and 150 MHz for 13C, Bruker Corporation, Bremen, Germany) in CDCl3 using tetramethylsilane as internal standard, d in ppm, and J in Hz; HR-ESI-MS were recorded on Waters LCT Premier XE timeof-flying mass spectrometer (Waters, Milford, CT, USA); and CD spectrum was measured on Biologic MOS-450 CD spectrometer (Bio-Logic, Grenoble, France). Column chromatography (CC) was performed on silica gel (200 – 300 mesh; Qingdao Marine Chemical Group, Co., Qingdao, China), octadecylsilane (30 – 50 mm; YMC Co. Ltd, Tokyo, Japan), and preparative HPLC [Hitachi L-7110 Pump, Hitachi L-7420 UV spectrophotometric detector at 210 nm, YMC C18 reversed phase column (YMC Co.) (5 mm, 10 mm £ 250 mm, flow rate: 2.0 ml/min)]. 3.2
Plant material
The roots of L. chuanxiong were bought from the medicine market (Shenyang, China) in October 2007 and identified by Prof. Jincai Lu, Shenyang Pharmaceutical University. A voucher specimen (No. SN002735) has been deposited in Shenyang Pharmaceutical University. 3.3 Exaction and isolation The powdered and air-dried roots (10.0 kg) of L. chuanxiong were refluxed three times
1241
using EtOH (95%, v/v) for 3 h to give a crude extract, which was suspended in H2O and extracted with petroleum ether (PE) to yield PE extract. A part of the PE fraction (95 g) was subjected to silica gel CC (400 g), gradiently eluted using PE/acetone to afford fractions 1–200. Fraction 26 (PE:acetone 100:1, 0.75 g) was subjected to preparative RP-HPLC (MeOH:H2O, 72:28; flow rate, 2.0 ml/min; wavelength, 210 nm) to yield 1 (99.5 mg, Rt 21.4 min). Fraction 45 (PE: acetone 100:4, 1.64 g) was rechromatographed over a silica gel column by eluting with PE/acetone to give fractions 1–150. Fractions 93 – 125 (PE:acetone 100:2, 0.086 g) were also purified by preparative RP-HPLC (MeOH:H2O, 70:30; flow rate 2.0 ml/min; wavelength 210 nm) to yield 2 (10.4 mg, Rt 31.5 min). 3.3.1
Chuanxiongdiolide A
Faint yellow oil (MeOH). ½a21 D þ 12.8 (c ¼ 0.78, MeOH). UV (MeOH) lmax: 222 nm. IR (KBr) vmax: 3478, 2955, 2870, 2285, 1749, 1661, 1465, 1053, 967, 748 cm21. CD (MeOH) spectral data: see Figure 4. 1H and 13C NMR spectral data: see Table 1. HR-ESI-MS: m/z 385.2375 [M þ H]þ (calcd for C24H33O4, 385.2379). 3.3.2 Chuanxiongdiolide B Faint yellow oil (MeOH). ½a21 D þ 19.2 (c ¼ 0.95, MeOH). UV (MeOH) lmax: 212 nm. IR (KBr) vmax: 3429, 3031, 2959, 2932, 2872, 2358, 1784, 1733, 1668, 1454, 1338, 1239, 1049, 953 cm21. CD (MeOH) spectral data: see Figure 4. 1H and 13C NMR spectral data: see Table 1. HR-ESIMS: m/z 401.2331 [M þ H]þ (calcd for C24H33O5, 401.2328). 3.4
Anti-BuChE activity assay
A mix of 25 ml DTNB [5,50 -dithio-bis (2nitrobenzoic acid)] (4.0 mM) and 25 ml enzyme solution (0.01 U/ml) was incubated at 378C in 96-well plates in the presence of 25-ml compounds (50 mM).
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J. Huang et al.
The reaction was started by adding 25 ml substrate solution (butyrylcholine chloride, 0.4 mM) and incubated for 40 min. Then, the absorbance of reaction product was tested at 405 nm (Bio-Rad Model 680 Automatic microplate reader, Thermo Fischer Scientific oy, Finland) [6].
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Inhibitory rate ð%Þ ¼ A405 ðenzymeÞ 2 A405 ðcompoundÞ £ 100: A405 ðenzymeÞ 2 A405 ðblankÞ Acknowledgments This work was financially supported by Key Projects of the National Science and Technology Pillar Program (No. 2012BAI30B02).
References [1] C.P. Miao, S.H. Wu, B.Z. Luo, J. Wang, and Y.W. Chen, Fitoterapia 81, 1088 (2010). [2] S.Y. Chor, A.Y. Hui, K.F. To, K.K. Chan, Y.Y. Go, H.L. Chan, W.K. Leung, and J.J. Sung, J. Ethnopharmacol. 100, 180 (2005). [3] L.S. Lim, P. Shen, and Y.H. Gong, Phytochemistry 67, 728 (2006). [4] N. Berova and K. Nakanishi, Circular Dichroism: Principles and Applications, 2nd ed. (Wiley-VCH, New York, 2000). [5] E. Giacobini, Pharmacol. Res. 50, 433 (2004). [6] R. Leo´n, C. Rı´os, M.C. Jose´, H. Oscar, B. Xavier, F.J. Luque, M.G. Lo´pez, A.G. Garcı´ab, and M. Villarroya, Bioorg. Med. Chem. 16, 7759 (2008).
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Two new phthalides with BuChE inhibitory activity from Ligusticum chuanxiong a
a
a
b
b
Jian Huang , Xiao-Qing Lu , Jin Lu , Guo-Yu Li , Hang-Yu Wang , a
c
ab
Li-Hong Li , Rui-Chao Lin & Jin-Hui Wang a
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, 110016, China b
School of Pharmacy, Shihezi University, Shihezi, 832002, China
c
National Institute for the Control of Pharmaceutical and Biological Products, Beijing, 100050, China Published online: 27 Nov 2013.
To cite this article: Jian Huang, Xiao-Qing Lu, Jin Lu, Guo-Yu Li, Hang-Yu Wang, Li-Hong Li, Rui-Chao Lin & Jin-Hui Wang (2013) Two new phthalides with BuChE inhibitory activity from Ligusticum chuanxiong, Journal of Asian Natural Products Research, 15:12, 1237-1242, DOI: 10.1080/10286020.2013.825610 To link to this article: http://dx.doi.org/10.1080/10286020.2013.825610
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Simon Fraser University] at 05:04 17 November 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2013 Vol. 15, No. 12, 1237–1242, http://dx.doi.org/10.1080/10286020.2013.825610
Two new phthalides with BuChE inhibitory activity from Ligusticum chuanxiong Jian Huanga, Xiao-Qing Lua, Jin Lua, Guo-Yu Lib, Hang-Yu Wangb*, Li-Hong Lia, Rui-Chao Linc and Jin-Hui Wangab* a
Downloaded by [Simon Fraser University] at 05:04 17 November 2014
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China; bSchool of Pharmacy, Shihezi University, Shihezi 832002, China; c National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China (Received 6 May 2013; final version received 11 July 2013) Two new phthalides, chuanxiongdiolides A (1) and B (2), were isolated from the roots of Ligusticum chuanxiong Hort. Their structures were established by UV, IR, 1D (1H, 13C) and 2D (HSQC, 1H – 1H COSY, HMBC, NOESY) NMR, and HR-ESI-MS methods, and their absolute configurations were assigned via circular dichroism exciton chirality. The two compounds showed different degrees of inhibitory effects against butyrylcholine esterase. Keywords: Ligusticum chuanxiong; chuanxiongdiolide A; chuanxiongdiolide B; NMR; CD; anti-BuChE activity
1.
Introduction
The root of Ligusticum chuanxiong, a popular traditional Chinese medicine, has been widely used in the treatment of headache, rheumatic arthralgia, menstrual disorders [1], and swelling as well as pain due to traumatic injuries and liver diseases [2]. Previous reports on the biological components revealed the presence of dimeric phthalides [3]. Being interested in finding more biologically active substances from this folk medicine, we further undertook the investigation to explore their phytochemical composition. As a result, two new phthalides, chuanxiongdiolide A (1) and chuanxiongdiolide B (2), were isolated from the roots of L. chuanxiong. In this paper, we describe the structural elucidation of the two new compounds and their anti-butyrylcholine esterase (BuChE) activity (Figure 1).
2.
Result and discussion
Compound 1 was obtained as faint yellow oil (MeOH) with the molecular formula C24H32O4 as determined by HR-ESI-MS at m/z 385.2375 [M þ H]þ, indicating nine degrees at unsaturation. The IR spectrum exhibited the maximum absorptions at 1749 cm21 (carbonyl) and 1661 cm21 (olefin moieties). The 1H NMR spectrum of 1 showed one olefinic proton at dH 7.29 (1H, d, J ¼ 6.6 Hz, H-70 ) and two oxygensubstituted methine signals at dH 4.88 (1H, dd, J ¼ 7.4, 3.0 Hz, H-3) and 5.13 (1H, dd, J ¼ 10.3, 2.7 Hz, H-3 0 ). Twenty-four C-atoms signals including 2 CH3, 10 CH2, 6 CH, and 6 quaternary C-atoms were evident from the analysis of the 1 H, 13C NMR, and HSQC spectra. The 13 C NMR spectrum showed the resonances of one 5-membered unsaturated lactone ring at dC 82.0, 126.5, 169.2, and 173.8,
*Corresponding authors. Email: [email protected]; [email protected] q 2013 Taylor & Francis
1238
J. Huang et al. 10
1
8 4
1
7 O
O
6'
3' 7a' O
10 9
9'
10'
O
7' O 3'
3a' 4' 8'
1'
11
3a
H 7a'
5' 8'
O
8
3
5 6
HO
3a'
7'
7a
O
7a 5' 4'
O
4 3
7
6'
Downloaded by [Simon Fraser University] at 05:04 17 November 2014
9
3a
5 6
11
9'
11' 1
2
10'
11'
Figure 1. Chemical structures of 1 and 2 isolated from L. chuanxiong.
and the resonances of one a,b-unsaturated ester at dC 135.6, 141.8, and 167.2. Comparison of the 1H and 13C NMR spectra of 1 (Table 1) with those of the known compound (3Z0 )-(3a0 R,60 R,3R,6R,7R)-3,8dihydro-6.60 ,7.3a0 -diligustilide [3] revealed that the resonances of C-30 at dC 150.4 and C-80 at dC 108.8 disappeared, and the replacements of an oxygen-substituted methine at dC 84.1 and a methylene at dC 31.8 in 1 were observed, indicating that the double bond between C-30 and C-80 was saturated. Further analysis of the correlations in the 2D NMR spectra including 1 H – 1H COSY, HMQC, and HMBC (Figure 2) led to the planar structure of 1 as shown in Figure 1. The relative configuration of 1 was elucidated on the basis of the analysis of the NOESY correlations and molecular dynamics calibration with minimum energy by using Chem3D (Figure 2). In the NOESY spectrum, the cross-peaks between H-70 and H-4, H-70 and H-5, H-7 and H-40 , H-3 and H-30 , and H-6 and H-7 were observed. It was shown that only the configuration shown in Figure 2 could give such stereo structure correlations. The absolute configuration of 1 was determined by the circular dichroism (CD) exciton chirality method [4]. In the CD spectrum, it showed a clearly positive
cotton effect at 230 nm and a negative cotton effect at 215 nm due to the transition interaction between two a,bunsaturated lactones, indicating a positive chirality for 1 (Figure 4). So, the absolute configuration of 1 was determined as shown, which is named as chuanxiongdiolide A. Compound 2 was also a faint yellow oil. The molecular formula C24H32O5 was determined by the HR-ESI-MS at m/z 401.2331 [M þ H] þ , indicating nine degrees of unsaturation. The IR spectrum showed absorptions at 3429 cm 21 (hydroxyl), 1784, 1733 cm21 (carbonyl), and 1668 cm21 (olefin moieties). The 13C NMR spectrum also exhibited 24 C-atom signals which consisted of 2 CH3, 10 CH2, 5 CH, and 7 quaternary C-atoms in combination with careful analysis of the data of 1H and HSQC spectra (Table 1). Moreover, it showed the resonances of one 5-membered unsaturated lactone ring at dC 82.5, 123.3, 166.5, and 172.8; one ester group at dC 178.7; and two pairs of olefinic carbons at dC 116.4, 125.7, 131.0, and 146.0. The above data were found to display the main characteristics of dimeric phthalides. Besides, the 13C NMR spectrum showed two oxygen-substituted methines at dC 65.7 and 82.5. Furthermore, analysis of the unsaturation degrees
Journal of Asian Natural Products Research Table 1.
1
H (600 MHz) and 13C (150 MHz) NMR spectral data for compounds 1 and 2 in CDCl3. 1
Downloaded by [Simon Fraser University] at 05:04 17 November 2014
Position
1239
dC
1 3 3a 4
173.8 82.0 169.2 21.4
5
28.3
6 7 7a 8
38.7 38.9 126.5 30.9
9
26.1
10 11 10 30 30 a 40
22.3 13.6 167.2 84.1 47.8 26.0
50
25.2
60 70 70 a 80
40.2 141.8 135.6 31.8
90
28.4
100 110
22.1 13.7
2
dH (J in Hz)
dC
4.88 (1H, dd, J ¼ 7.4, 3.0) 2.07 (2H, m) 1.54 (1H, m) 1.85 (1H, m) 2.43 (1H, m) 2.94 (1H, br. d, J ¼ 8.8) 1.40 (1H, m) 1.86 (1H, m) 1.22(1H, m) 1.31(1H, m) 1.37(2H, m) 0.88 (3H, t, J ¼ 7.2)
1.39 (1H, m) 1.79 (1H, m) 1.29 (1H, m) 1.87 (1H, m) 2.97 (1H, dd, J ¼ 6.6, 2.4) 7.29 (1H, d, J ¼ 6.6) 1.57 (1H, m) 1.78 (1H, m) 1.48 (1H, m) 1.60 (1H, m) 1.31 (2H, m) 0.92 (3H, t, J ¼ 7.2)
O O HMBC
131.0 125.7 53.9 24.9
22.2 13.7 172.8 82.5 166.5 19.3 25.5 65.7 45.8 123.3 32.4
2.25 (1H, m) 2.47 (1H, m) 2.11 (1H, m) 2.27 (1H, m) 5.83 (1H, d, 9.7) 5.94 (1H, d, 9.7) 2.25 (1H, m) 2.31 (1H, m) 1.50 (2H, m) 1.33 (2H, m) 0.91 (3H, t, 7.2) 4.76 (1H, br.d, 6.2) 2.32 (1H, m) 2.38 (1H, m) 1.92 (1H, m) 2.22 (1H, m) 4.26 (1H, br. s) 2.98 (1H, br. s)
26.6
1.47 (1H, m) 1.88 (1H, m) 1.39 (2H, m)
22.5 13.9
1.35 (2H, m) 0.91 (3H, t, 7.2)
NMR spectra including 1H – 1H COSY, HMQC, and HMBC (Figure 3) deduced the gross structure of 2 as shown in
O
O
27.8
28.5
5.13 (1H, dd, J ¼ 10.3, 2.7)
inferred that the dimer was formed through one single bond of C-atoms. Careful analysis of the correlations in the 2D
178.7 146.0 116.4 18.8
dH (J in Hz)
NOESY
Figure 2. Key HMBC and NOESY correlations of 1.
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J. Huang et al. O O
HO
H
O O
HMBC
NOESY
Figure 1. Especially in the HMBC spectrum, the correlations from H-70 to C-3a and C-1, H-7 to C-70 , and H-60 to C7a verified that the two monophthalides were connected at C-7a and C-70 . The relative configuration of 2 was also determined via a NOESY experiment and molecular dynamics calibration with minimum energy by using the Chem3D (Figure 3), in which the correlations between H-30 and H-40 (dH 2.32), H-40 (dH 2.32) and H-50 (dH 1.92), H-50 (dH 1.92) and H-60 , and H-60 and H-70 were observed. Only the configuration shown in Figure 4 could give such stereo structure corre-
lations, indicating that the hydrogen bond between 60 -OH and 1ZCvO rendered the molecule in such stable configuration. In addition, the absolute configuration of 2 was determined by the CD exciton chirality method [4]. It showed a clearly positive cotton effect at 230 nm and a negative cotton effect at 215 nm due to the transition interaction between two excitons (Figure 4); so, the absolute configuration of 2 was determined as shown in Figure 1, which is named as chuanxiongdiolide B. BuChE was closely related with neurodegenerative diseases [5]. Therefore,
(+) (+)
∆ (M–1 cm–1)
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Figure 3. Key HMBC and NOESY correlations of 2.
3 2.5 2 1.5 1 0.5 0 –0.5 –1 –1.5 –2 190
2
1
Compound 1 Compound 2 240
290 Wavelength(nm)
340
Figure 4. CD spectra and exciton chirality of compounds 1 and 2.
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Journal of Asian Natural Products Research anti-BuChE activity was evaluated for 1 and 2 in the concentration of 50 mM. The results showed that inhibitory rates of 1 and 2 were 63.0% and 21.7%, respectively, with the tetraisopropyl pyrophosphoramide (37.7%) as the positive control.
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3.
Experimental
3.1 General experimental procedures UV spectra were measured using the Shimadzu UV-2401 spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra were run on IRAffinity-1 FTIR spectrometer (Shimadzu). NMR spectra were obtained on Bruker AV-600 spectrometer (600 MHz for 1H and 150 MHz for 13C, Bruker Corporation, Bremen, Germany) in CDCl3 using tetramethylsilane as internal standard, d in ppm, and J in Hz; HR-ESI-MS were recorded on Waters LCT Premier XE timeof-flying mass spectrometer (Waters, Milford, CT, USA); and CD spectrum was measured on Biologic MOS-450 CD spectrometer (Bio-Logic, Grenoble, France). Column chromatography (CC) was performed on silica gel (200 – 300 mesh; Qingdao Marine Chemical Group, Co., Qingdao, China), octadecylsilane (30 – 50 mm; YMC Co. Ltd, Tokyo, Japan), and preparative HPLC [Hitachi L-7110 Pump, Hitachi L-7420 UV spectrophotometric detector at 210 nm, YMC C18 reversed phase column (YMC Co.) (5 mm, 10 mm £ 250 mm, flow rate: 2.0 ml/min)]. 3.2
Plant material
The roots of L. chuanxiong were bought from the medicine market (Shenyang, China) in October 2007 and identified by Prof. Jincai Lu, Shenyang Pharmaceutical University. A voucher specimen (No. SN002735) has been deposited in Shenyang Pharmaceutical University. 3.3 Exaction and isolation The powdered and air-dried roots (10.0 kg) of L. chuanxiong were refluxed three times
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using EtOH (95%, v/v) for 3 h to give a crude extract, which was suspended in H2O and extracted with petroleum ether (PE) to yield PE extract. A part of the PE fraction (95 g) was subjected to silica gel CC (400 g), gradiently eluted using PE/acetone to afford fractions 1–200. Fraction 26 (PE:acetone 100:1, 0.75 g) was subjected to preparative RP-HPLC (MeOH:H2O, 72:28; flow rate, 2.0 ml/min; wavelength, 210 nm) to yield 1 (99.5 mg, Rt 21.4 min). Fraction 45 (PE: acetone 100:4, 1.64 g) was rechromatographed over a silica gel column by eluting with PE/acetone to give fractions 1–150. Fractions 93 – 125 (PE:acetone 100:2, 0.086 g) were also purified by preparative RP-HPLC (MeOH:H2O, 70:30; flow rate 2.0 ml/min; wavelength 210 nm) to yield 2 (10.4 mg, Rt 31.5 min). 3.3.1
Chuanxiongdiolide A
Faint yellow oil (MeOH). ½a21 D þ 12.8 (c ¼ 0.78, MeOH). UV (MeOH) lmax: 222 nm. IR (KBr) vmax: 3478, 2955, 2870, 2285, 1749, 1661, 1465, 1053, 967, 748 cm21. CD (MeOH) spectral data: see Figure 4. 1H and 13C NMR spectral data: see Table 1. HR-ESI-MS: m/z 385.2375 [M þ H]þ (calcd for C24H33O4, 385.2379). 3.3.2 Chuanxiongdiolide B Faint yellow oil (MeOH). ½a21 D þ 19.2 (c ¼ 0.95, MeOH). UV (MeOH) lmax: 212 nm. IR (KBr) vmax: 3429, 3031, 2959, 2932, 2872, 2358, 1784, 1733, 1668, 1454, 1338, 1239, 1049, 953 cm21. CD (MeOH) spectral data: see Figure 4. 1H and 13C NMR spectral data: see Table 1. HR-ESIMS: m/z 401.2331 [M þ H]þ (calcd for C24H33O5, 401.2328). 3.4
Anti-BuChE activity assay
A mix of 25 ml DTNB [5,50 -dithio-bis (2nitrobenzoic acid)] (4.0 mM) and 25 ml enzyme solution (0.01 U/ml) was incubated at 378C in 96-well plates in the presence of 25-ml compounds (50 mM).
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The reaction was started by adding 25 ml substrate solution (butyrylcholine chloride, 0.4 mM) and incubated for 40 min. Then, the absorbance of reaction product was tested at 405 nm (Bio-Rad Model 680 Automatic microplate reader, Thermo Fischer Scientific oy, Finland) [6].
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Inhibitory rate ð%Þ ¼ A405 ðenzymeÞ 2 A405 ðcompoundÞ £ 100: A405 ðenzymeÞ 2 A405 ðblankÞ Acknowledgments This work was financially supported by Key Projects of the National Science and Technology Pillar Program (No. 2012BAI30B02).
References [1] C.P. Miao, S.H. Wu, B.Z. Luo, J. Wang, and Y.W. Chen, Fitoterapia 81, 1088 (2010). [2] S.Y. Chor, A.Y. Hui, K.F. To, K.K. Chan, Y.Y. Go, H.L. Chan, W.K. Leung, and J.J. Sung, J. Ethnopharmacol. 100, 180 (2005). [3] L.S. Lim, P. Shen, and Y.H. Gong, Phytochemistry 67, 728 (2006). [4] N. Berova and K. Nakanishi, Circular Dichroism: Principles and Applications, 2nd ed. (Wiley-VCH, New York, 2000). [5] E. Giacobini, Pharmacol. Res. 50, 433 (2004). [6] R. Leo´n, C. Rı´os, M.C. Jose´, H. Oscar, B. Xavier, F.J. Luque, M.G. Lo´pez, A.G. Garcı´ab, and M. Villarroya, Bioorg. Med. Chem. 16, 7759 (2008).
